We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.